The administration of spironolactone (100 mg/kg, i.v.) to dogs causes a decrease of 40-50 percent within 4 to 5 hr in the adrenal or testicular content of the activities of steroid hydroxylases, thereby resulting in a 40-50 percent lowering of the plasma concentration of testosterone, estradiol and cortisol. The loss of the steroid hydroxylase activities by spironolactone stems from a specific destruction of the heme of cytochrome P-450 resulting in the rapid degradation of its apoproteins. Studies with several analogues of spironolactone have shown that the -SH group at the carbon-7 position of the steroid moiety is required for the loss of adrenal or testicular steroid hydroxylases. In vitro experiments with microsomal suspensions have indicated: 1) that spironolactone can be activated to cause a destruction of P-450 and its heme without a loss of the apoprotein, 2) that the activation requires NADPH, O2 and an active P-450 and 3) that the loss of P-450 can be prevented by addition of progesterone or by the conversion of P-450 to its inactive form P-420.